Oxidative stress plays a major role in chlorpromazine-induced cholestasis in human HepaRG cells

Authors

  • Sébastien Anthérieu,

    1. Inserm UMR991, Liver Metabolisms and Cancer, Rennes, France
    2. Université de Rennes 1, Rennes, France
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    • These authors contributed equally to this work.

  • Pamela Bachour-El Azzi,

    1. Inserm UMR991, Liver Metabolisms and Cancer, Rennes, France
    2. Université de Rennes 1, Rennes, France
    3. Université Libanaise, EDST-PRASE and EDST-AZM-center-LBA3B, Lebanon
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    • These authors contributed equally to this work.

  • Julie Dumont,

    1. Inserm UMR991, Liver Metabolisms and Cancer, Rennes, France
    2. Université de Rennes 1, Rennes, France
    Current affiliation:
    1. Inserm UMR 744, Université Lille Nord de France, Institut Pasteur, Lille, France
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  • Ziad Abdel-Razzak,

    1. Université Libanaise, EDST-PRASE and EDST-AZM-center-LBA3B, Lebanon
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  • Christiane Guguen-Guillouzo,

    1. Inserm UMR991, Liver Metabolisms and Cancer, Rennes, France
    2. Université de Rennes 1, Rennes, France
    3. Biopredic International, St Grégoire, France
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  • Bernard Fromenty,

    1. Inserm UMR991, Liver Metabolisms and Cancer, Rennes, France
    2. Université de Rennes 1, Rennes, France
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  • Marie-Anne Robin,

    1. Inserm UMR991, Liver Metabolisms and Cancer, Rennes, France
    2. Université de Rennes 1, Rennes, France
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  • André Guillouzo

    Corresponding author
    1. Inserm UMR991, Liver Metabolisms and Cancer, Rennes, France
    2. Université de Rennes 1, Rennes, France
    • Inserm UMR 991, Faculté des Sciences Pharmaceutiques et Biologiques, 35043 Rennes Cedex, France===

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    • fax: +33 223235385


  • Potential conflict of interest: Nothing to report.

Abstract

Drugs induce cholestasis by diverse and still poorly understood mechanisms in humans. Early hepatic effects of chlorpromazine (CPZ), a neuroleptic drug known for years to induce intrahepatic cholestasis, were investigated using the differentiated human hepatoma HepaRG cells. Generation of reactive oxygen species (ROS) was detected as early as 15 minutes after CPZ treatment and was associated with an altered mitochondrial membrane potential and disruption of the pericanalicular distribution of F-actin. Inhibition of [3H]-taurocholic acid efflux was observed after 30 minutes and was mostly prevented by N-acetyl cysteine (NAC) cotreatment, indicating a major role of oxidative stress in CPZ-induced bile acid (BA) accumulation. Moreover, 24-hour treatment with CPZ decreased messenger RNA (mRNA) expression of the two main canalicular bile transporters, bile salt export pump (BSEP) and multidrug resistance protein 3 (MDR3). Additional CPZ effects included inhibition of Na+-dependent taurocholic cotransporting polypeptide (NTCP) expression and activity, multidrug resistance-associated protein 4 (MRP4) overexpression and CYP8B1 inhibition that are involved in BA uptake, basolateral transport, and BA synthesis, respectively. These latter events likely represent hepatoprotective responses which aim to reduce intrahepatic accumulation of toxic BA. Compared to CPZ effects, overloading of HepaRG cells with high concentrations of cholic and chenodeoxycholic acids induced a delayed oxidative stress and, similarly, after 24 hours it down-regulated BSEP and MDR3 in parallel to a decrease of NTCP and CYP8B1 and an increase of MRP4. By contrast, low BA concentrations up-regulated BSEP and MDR3 in the absence of oxidative stress. Conclusion: These data provide evidence that, among other mechanisms, oxidative stress plays a major role as both a primary causal and an aggravating factor in the early CPZ-induced intrahepatic cholestasis in human hepatocytes. (HEPATOLOGY 2013)

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